| Globulin is one of the major protein fractions of Juglans mandshurica Maxim.(JMM) kernel which is worth further exploiting and utilizing. This paper studied Juglans mandshurica Maxim. kernel globulin(JMG) which was extracted via the optimized method of Osborne and Juglans mandshurica Maxim kernel defatted meal as raw materials. Investigated the affects of extraction solvents, the ratio of material to solvent, and the p H of extraction for Juglans mandshurica Maxim. kernel protein globulin extraction efficiency and purity by single factor experiments. Finally by orthogonal test determined the optimal extraction conditions were follows: extraction solvents is PBS, the ratio of material to solvent is 1:10, and the p H of extraction is 7.5. It was proved that the protein content of JMG reached 80.71%. Then the raw globulin was purified. Firstly, the solution of JMG was applied to a column of anion-exchange with 50 m M KH2P04‐Na2HP04 buffer(p H 7.5). The sample was eluted with a linear gradient of Na Cl(0-0.6 mol/L) in the buffer.;The total volume of the gradient solution was 15 times column volume and fractions 5m L were collected with a flow rate of 1.0 m L/min. Secondly, the sample obtained above was applied to a Hi Prep16/60 Sephacryl S-200 HR column equilibrated with the buffer(50mmol/L KH2P04‐Na2HP04 buffer(p H 7.5) containing 0.2 mol/L Na Cl), loading volume was 2.0m L.The column was eluted with the equilibrated buffer at a flow rate of 0.2 m L/min. Under reducing conditions, JSG was composed of two major polypeptides with molecular masses of ~23 and ~20 k Da. In the absence of a reducing agent(β-mercaptoethanol), JSG showed a single protein band in the region of ~40 k Da. MALDI-TOF/TOF MS analysis indicated that the polypeptides were distinct from each other and that their amino acid sequence was highly similar to the 11 S globulin from Juglans regia in the NCBI database.In this paper,the properties of JSG was characterization. Amino acid composition analysis showed that JSG contained all essential amino acids, Lysine was the first limiting amino acid of JSG. Differential scanning calorimetry revealed a single endothermic peak. The denaturation temperature was 96.83 °C, and the enthalpy value was 12.07 J/g. Circular dichroism spectra showed that the secondary structure of JSG consisted of β-sheets and α-helices. A red shift was also observed in the fluorescence spectra with the denaturant guanidine hydrochloride(Gdn HCl) or urea; Gdn HCl was proven to be a stronger denaturant than urea. When p H was greater than 10 or less than 5, the peak intensity rapidly reduced with pronounced redshift.To provide theoretical basis for its application in food processing, its functional properties were fully analyzed by compared with soybean protein isolate(SPI). The essential amino acids contents of JMG met the Adult Standard recommended by FAO/WTO. Differential Scanning Calorimetry(DSC)spectrogram existed two endothermic peaks, showing that the denaturation temperatures of globulin powder were 98.08 ℃and 155.33 ℃, the enthalpy value were 13.06 J/g,764.80 J/g, respectively. Fourier infrared spectrum(FT-IR)showed the secondary structure was given priority to with alpha helix. Except the foamability and water absorption were significantly lower than SPI, other functional properties of JMG were corresponded to SPI. |
PDF Full Text Request